The present invention relates generally to genetically engineered organisms useful for screening pharmaceutical compounds having antimicrobial capability and, more particularly, to recombinant organisms in which the expression of an RNA polymerase specificity factor can be regulated. The present invention is more specifically directed to recombinant bacterial strains in which the expression of RNA polymerase "sgr" subunit can be regulated by the addition of an exogenous effector molecule. In particular, the present invention relates to a new recombinant staphylococcal bacterial strain having an engineered inducer-responsive gene for regulating expression of RNA polymerase a subunit and methods for utilizing the same in high throughput screening to detect antimicrobial compounds pharmaceutically useful against staphylococcal bacteria.
Numerous pathogenic organisms, such as Staphylococcus aureus (xe2x80x9cS. aureusxe2x80x9d), are responsible for infectious disease and health-related problems in humans and other animals throughout the United States and the world. As treatments are developed for combating a particular organism, such as treatments incorporating newly developed antibiotics and chemical compounds effective at eliminating existing strains of a particular organism, newer strains of such organisms emerge which are resistant to the existing treatments. Accordingly, there remains a constant need for the development of new ways for pharmaceutically combating pathogenic organisms.
Methods for combating an organism by interfering with genetic processes essential to survival and growth of the organism are becoming of increasing interest. In particular, researchers are directing their attention to chemical compounds which interfere with genetic transcription processes required for growth or continued existence of pathogenic organisms.
The expression of genetic information in an organism ultimately occurs via proteins, and particularly by enzymatic proteins which catalyze metabolic reactions. The flow of genetic information from DNA to protein occurs generally in two steps, termed xe2x80x9ctranscriptionxe2x80x9d and xe2x80x9ctranslationxe2x80x9d. Transcription is the first step in the flow of genetic information, whereby DNA-encoded genetic information is copied into RNA. The further conversion of RNA into protein occurs by the process of translation. A xe2x80x9cgenexe2x80x9d is broadly a region of DNA which encodes one protein, and the transcription-translation process of forming that protein is termed xe2x80x9cexpressionxe2x80x9d of the gene.
In particular, transcription involves the synthesis of an RNA chain representing one strand of a DNA duplex. Importantly, RNA synthesis is catalyzed by an enzyme known as RNA polymerase. Transcription begins when RNA polymerase binds to a special region at the beginning of a DNA gene known as a promoter site.
RNA polymerase is generally comprised of two components, a core enzyme and a specificity factor. The specificity factor is particularly concerned with recognition and binding of the enzyme to the promoter region of a particular gene or set of genes on a DNA template. While the core enzyme component of RNA polymerase has the ability to synthesize RNA on a DNA template, it cannot initiate transcription at the promoter site without an associated specificity factor. The function of the specificity factor, thus, is to insure that RNA polymerase binds in a stable manner to DNA only at appropriate promoter sites. Consequently, the specificity factor directs binding of RNA polymerase at cognate promoter sequences, thereby initiating expression of only those selected genes incorporating the cognate promoter sequences.
Accordingly, the expression of a particular gene or set of genes can be controlled by regulating production of a corresponding specificity factor. In particular, the expression of a particular gene or set of genes can be inhibited by blocking the associated specificity factor, thereby preventing the binding of RNA polymerase to the gene promoter sequences. As a result, an attractive target for the treatment of a pathogenic organism would be the discovery of chemical agents which block an RNA polymerase specificity factor required for the expression of a gene or set of genes essential for continued existence of the organism.
One particular pathogenic organism of concern is the bacterium S. aureus, which is an opportunistic human pathogen and is the primary cause of nosocomial bacterial infections in the United States. S. aureus is associated with a number of life threatening systemic illnesses, such as bacteremia/sepsis, toxic shock syndrome and toxic epidermal necrolysis, as well as common bacterial infections of the skin. The recent emergence of methicillin-resistant and vancomycin-resistant strains of S. aureus has focused renewed attention on the need for development of new classes of antibiotics to combat such bacterial strains. A promising way of pharmaceutically combating bacterial strains, including S. aureus and other staphylococcal strains, is to interfere with genetic transcription processes relating to growth of the bacteria.
As in other eubacteria, the RNA polymerase of S. aureus is composed of two components, the core enzyme and a specificity factor. The core enzyme has a subunit composition of (xcex12xcex2xcex2xe2x80x2. The xcex2 and xcex2xe2x80x2 subunits together make up the catalytic center of the enzyme while the xcex1 subunit is required for assembly of the core enzyme, as well as other functions in promoter recognition.
The specificity factor in S. aureus is one of several "sgr" subunits or factors. The principle "sgr" factor, encoded and expressed in S. aureus by the chromosomal gene plaC, is required for the expression of essential housekeeping genes required for bacterial growth. As a result, this "sgr" factor is an attractive target for the discovery of chemical agents which possess antibacterial properties by binding with or blocking the "sgr" factor function, since the blocking of or interfering with the "sgr" factor function can prevent expression of these housekeeping genes and retard or prevent growth of the bacteria.
U.S. Pat. Nos. 5,585,277 and 5,679,582 to Bowie et al. disclose methods for screening chemical compounds for potential pharmaceutical or antimicrobial effectiveness. In particular, these patents teach methods for identifying possible therapeutic ligands which bind to target proteins. The methods of these patents may be useful in affinity-based assays for the initial identification of chemical compounds as in vitro inhibitors of an RNA polymerase and more specifically an RNA polymerase specificity factor, such as the primary "sgr" subunit in staphylococcal bacteria. However, there remains a need in the art for screening methods, and recombinant organisms useful therein, which monitor the in vivo effects of chemical compounds on the growth and/or survival of a target organism. Specifically, there remains a need in the art for recombinant organisms, and production methods therefor, in which the production of an RNA polymerase specificity factor which encodes a gene essential for growth or continued existence of the organism can be specifically controlled for use in screening processes. In particular, there remains a need in the art for a staphylococcal bacterial strain in which the production of RNA polymerase "sgr" subunit can be specifically controlled for use in screening methods. Moreover, there is a need in the art for specific methods and compositions that allow the identification of antimicrobial agents which interact with and/or modify the function of the RNA polymerase "sgr" subunit thereby inhibiting bacterial growth.
Accordingly, it is an object of the present invention to provide an organism in which the production of an RNA polymerase specificity factor can be controlled.
It is a further object of the present invention to provide a recombinant organism having a regulatable gene which encodes an RNA polymerase specificity factor required for expression of a gene or set of genes essential for growth or continued existence of the organism.
It is still a further object of the present invention to provide a method for producing such a recombinant organism.
It is another object of the present invention to provide a staphylococcal bacterial strain in which the production of RNA polymerase a subunit can be controlled.
It is still another object of the present invention to provide an engineered staphylococcal bacterial strain which allows the identification of antimicrobial agents that interact with the function of the RNA polymerase "sgr" subunit thereof.
Yet another object of the present invention is to provide a method for high throughput screening to detect candidate antimicrobial compounds useful against certain organisms, and against staphylococcal bacterial strains in particular.
Still another object of the present invention is to provide a method of profiling drug susceptibilities of S. aureus carrying an inducible gene for expressing RNA polymerase "sgr" factor.
According to the present invention then, a recombinant organism is provided which has a regulatable gene for encoding an RNA polymerase specificity factor required for expression of a selected gene essential for growth of the organism. In particular, the recombinant organism may be a recombinant bacterial strain wherein the regulatable gene is an inducer-responsive gene including an operator site to which a repressor is capable of binding either completely or incompletely thereby to repress gene transcription of the RNA polymerase specificity factor.
More specifically, the present invention is directed to a recombinant staphylococcal bacterial strain which has a regulatable gene for encoding staphylococcal RNA polymerase "sgr" subunit required for expression of selected housekeeping genes relating to growth of the staphylococcal bacterial strain. In particular, the regulatable gene may include a lac operator with which a lacl-encoded repressor is capable of binding either completely or incompletely thereby to repress gene transcription of the RNA polymerase "sgr" subunit. The regulatable gene is preferably an IPTG responsive plaC allele, and the recombinant staphylococcal bacterial strain is preferably a S. aureus strain.
The present invention is also directed to a method for producing a recombinant organism having a regulatable gene for encoding an RNA polymerase specificity factor required for expression of a selected gene essential for growth or continued existence of the organism. The method includes the steps of identifying a wild-type gene encoding the RNA polymerase specificity factor, generating target gene fragments thereof which lack native transcriptional control sequences, and ligating the target gene fragements into an appropriate integrative vector at a location downstream of an inducible promoter. The integrative vector is then integrated into a chromosome of a wild-type organism to produce the recombinant organism. The integrative vector may specifically include a repressor-encoding gene, a resistance determinant, a multicloning site for insertion of the target gene fragment and a transcription terminator.
In particular, the method is directed to producing a recombinant staphylococcal bacterial strain, such as a strain of S. aureus, which has a regulatable gene for encoding RNA polymerase "sgr" subunit. The wild-type gene is preferably plaC, and the integrative vector preferably is a plasmid which includes a lad gene for encoding a repressor, a multicloning site preferably including an Srfl site for insertion of the target gene fragments, a transcription terminator, and a chloramphenicol resistance determinant.
A further embodiment of the invention relates to a method for high throughput screening to identify candidate pharmaceutical compounds that are effective against an organism, such as staphylococcal bacterial strains and S. aureus in particular. In particular, the method identifies candidate pharmaceutical compounds that target an RNA polymerase specificity factor, such as the "sgr" factor in S. aureus, required for expression of at least one gene essential for continued existence, e.g., growth and/or viability, of the organism. The method comprises the steps of preparing a plurality of cultures, contacting each of the cultures with a test compound, and monitoring the cultures to detect results indicating that the test compound targets the RNA polymerase specificity factor. The step of preparing a plurality of cultures includes preparing cultures of a first type and a second type. The first type of cultures includes a recombinant strain of the organism that has a regulatable gene for encoding the RNA polymerase specificity factor, whereas the second type of cultures include a wild-type strain of the organism that has a wild-type gene for encoding the RNA polymerase specificity factor. The regulatable gene may be one which is regulated by various methods, such as the use of repressor and inducer molecules as known in the art. The regulatable gene is expressed at basal levels in some of the cultures of the first type, and the regulatable gene is expressed at above basal levels in others of the cultures of the first type as a result of exposure of some of the cultures of the first type to an exogenous effector molecule, such as a gene inducing agent or a repressor molecule initiator as determined by the regulatory condition of the regulatable gene. Some of the cultures of the second type are also exposed to the exogenous effector molecule.
When a gene inducing agent is added as the exogenous effector molecule, the method may include the further step of adding the gene inducing agent in a sub-maximal concentration to those cultures of the first type that were not exposed to the gene inducing agent, if necessary to induce basal level expression of those cultures. Prior to the step of contacting the cultures with the test compound, the cultures are preferably incubated for a selected interval of time under conditions sufficient for growth of each culture to approach a target density. Additionally, after the step of contacting the cultures with the test compound, the cultures are preferably incubated under conditions that support growth of the cultures.
The cultures may be organized into sets and groups which may be organized in an array of columns and rows. Each group is preferably contacted with a different concentration of test compound. The method may be repeated for a plurality of test compounds.
The step of monitoring may be accomplished by measuring the growth of the cultures wherein a test compound that is a candidate pharmaceutical compound inhibits growth of cultures of the first type in which the gene is expressed at basal levels. Alternatively, a candidate pharmaceutical compound may be identified as one that inhibits RNA synthesis in cultures of the first type in which the regulatable gene is expressed at basal levels.
The present invention also relates to a method for high throughput screening to identify candidate pharmaceutical compounds that target an RNA polymerase specificity factor, comprising the steps of preparing a recombinant strain having a regulatable gene for encoding the RNA polymerase specificity factor, growing the strain, isolating and purifiying the RNA polymerase specificity factor, contacting the RNA polymerase specificity factor with a test compound and detecting whether the test compound binds to said RNA polymerase specificity factor.
These and other objects of the present invention will become more readily appreciated and understood from a consideration of the following detailed description of the exemplary embodiments of the present invention when taken together with the accompanying drawings, in which: